As a technique for obtaining information about tumor in the body by sampling blood, circulating tumor DNA (ctDNA), which is cell free DNA (cfDNA) released in blood from a dead cancer cell, has recently been used. This ctDNA is believed to be a carrier for transferring genetic information about solid tumor to a peripheral blood vessel, and it is expected that the use of ctDNA facilitates analyses of hereditary tumor heterogeneity (e.g., cancer cell evolution caused by the disease course). The size of cfDNA is 170 base pairs on average and its half-life is 16.5 minutes. One to several thousand genomic ctDNAs are contained in one milliliter of blood.
A wide variety of techniques have been developed for detecting this ctDNA, while digital PCR and its related techniques, particularly massively parallel DNA sequencers, so-called next-generation sequencers, are leading techniques. However, the disadvantage of such next-generation sequencers is read errors that frequently occur, and the number of erroneous constants and false positive increases, as the number of regions for which base sequences should be determined increases. Another disadvantage is that there is a step for amplifying the entire DNA to be analyzed at a template preparation stage for massively parallel sequencing, and thereby the final sequence reads do not reflect the first proportion of DNA molecules. The number of reads normally exceeds the number of DNA molecules to be analyzed, which in turn influences the measurement of alleles in mutations.
A technique used for solving the abovementioned advantages is a barcode sequence (Casbon, J. A., Osborne, R. J., Brenner, S. and Lichtenstein, C. P. 2011, A method for counting PCR template molecules with application to next-generation sequencing. Nucleic Acids Res., 39, e81. Kinde, I., Wu, J., Papadopoulos, N., Kinzler, K. W. and Vogelstein, B. 2011, Detection and quantification of rare mutations with massively parallel sequencing. Proc. Natl. Acad. Sci. USA, 108, 9530-9535). This method enables to label DNA fragments with a random sequence having 10-15 bases in many cases, distinguish reads originated from individual molecules and then group reads originated from each molecule. In other words, DNA sequencing of high quality can be provided by making a consensus of reads, so that the number of sequenced molecules can be counted.